The Roles of Polymerase Gamma Accessory Subunit Gene Mutations in Human Disease.

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DESCRIPTION (provided by applicant): The human mitochondrial DNA (mtDNA) polymerase, (Polg) is composed of two subunits encoded by two nuclear genes: 1. the POLG gene encoding the catalytic subunit, p140 and 2. The POLG2 gene encoding the homodimeric accessory subunit, p55. Polg is the sole cellular mtDNA polymerase for mtDNA replication and repair. Mitochondrial diseases, including those linked to POLG2 mutations, are devastating disorders that comprise a continuum of overlapping phenotypes and the age of onset of these diseases range from early childhood to late adulthood. Currently there are no cures for these diseases. To date, al reported POLG2 mutations associated with human mitochondrial disease occur as heterozygous mutations. In vivo dysfunction associated with POLG2 mutations likely results from dominant negative p55 variants that negatively affect or block the function of wild-type gene products, namely p140 and p55. Major barriers to this field of research include the lack of characterization of heterodimeric p55 variants and the limitations of biochemical approaches that exclude analysis of pathogenic alleles. Another barrier in the field is the absence of cell models harboring POLG2 mutations to determine and characterize cellular mechanisms of respiratory dysfunctions. During the K99/Mentored phase Dr. Young's aim is to determine dominant negative mechanisms of POLG2 mutations. Dr. Young has developed and will exploit p55 heterodimeric variants to determine dominant negative mechanisms in vitro. In alliance with heterodimeric studies Dr. Young has developed stable human cell lines with POLG2 mutations to determine dominant negative mechanisms in vivo. Dr. Young will receive structured training in qualitative analysis of mtDNA deletions and quantitative measurements of mtDNA copy number to investigate cellular dysfunctions of mtDNA maintenance linked to POLG2 mutations. In the R00/Independent phase of this award Dr. Young will study prospectively human stable cell line models to address the second aim of determining mechanisms of respiratory defects associated with mitochondrial disease mutations. He will exploit already developed human cell lines harboring POLG2 mutations and develop new cell models to determine mechanisms of respiration deficiency. These studies are essential to fundamental research and determining the biological mechanisms of the mitochondrion that respond to disease mutations. The applicant's long-term objective is targeted at exposure research and determining how drugs and toxins affect mtDNA maintenance and respiration in wild-type and disease states, so called mitochondrion-environment interactions. The principle investigator, Dr. Young, is well suited to carry out the proposed research plans based on his prior training in the mitochondrial research field. His prior training includes yeast mitochondrial genetics, development of human stable cell lines harboring POLG2 mutations, and biochemical characterization of Polg variants. The proposed training and career development will enable Dr. Young to become an independent biomedical research scientist and prepare him for a leadership role managing a mitochondrial research laboratory.